Host-Frankia specificity within the Casuarinaceae

Summary Fifteen species from three genera of the Casuarinaceae were inoculated with suspensions ofFrankia prepared from single nodule-lobes collected from different species and genera within the Casuarinaceae. Host-endophyte specificity was expressed mainly at the generic level. There was marked cross-inoculation within Casuarina and little nodulation ofCasuarina species from Allocasuarina sources with the exception of 3 sources ofFrankia fromA. torulosa which showed a high tendency to nodulateCasuarina species. Few sources from Casuarina nodulated species of Allocasuarina and while cross-inoculation within Allocasuarina was frequent it was less marked than within Casuarina. SomeFrankia inocula had wider host ranges than others, nodulating outside the genus or series of origin. It was not possible to determine if these apparent wider ranges in host spectra reflected genotypic differences betweenFrankia or were associated with the presence of more than oneFrankia strain in some inocula.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Host plant growth response to inoculation withFrankia

Optimum growth conditions and inoculation regimes were determined for severalFrankia strains isolated from both Alnus and Casuarina host plants. Growth conditions were estabilished that allowed a reduction in generation time to less than 15 hours for certain Alnus derivedFrankia. Differences in plant growth response were observed with differing inoculum levels and soil mixtures. Elite strains of Alnus derivedFrankia were isolated that elicited similar growth reponses in allAlnus species tested; however, differences were observed betweenFrankia strains and plant growth response of variousCasuarina species tested.     

Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

The production and utilisation of monoclonal antibodies for identification of a Frankia strain utilised as inoculum for Casuarina equisetifolia

Monoclonal antibodies were raised against Frankia 0RS020607, a strain isolated originally by H.G. Diem from nodules of Casuarina equisetifolia from Senegal. One of these antibodies, mAb8C5, was shown by ELISA to have high, but not absolute specificity for 0RS020607. This antibody was employed to investigate the mobility and persistence of 0RS020607 in plantations of C. equisetifolia. Seedlings were inoculated in pots of sand in a forest nursery with 0RS020607, with local crushed nodule suspensions or were left uninoculated. They were planted out after 5 months in experimental plots on a moderately fertile black soil site and on a low organic, oxidised red soil site. Compared with crushed nodule inoculated seedlings or uninoculated controls, growth of seedlings at transplant was improved by inoculation with Frankia 0RS020607. However, 4 years after transplant to experimental plots, the growth of trees receiving different treatments was similar. The possibility that movement of ORS 020607 between treatment plots contributed to new nodulation and enhanced growth of uninoculated trees was tested using mAb8C5 in ELISA of Frankia mycelium, extracted from the nodules of trees of the three treatments. No significant differences in reactivity were detected between nodules from uninoculated and 0RS020607 inoculated trees at either the black or the red soil sites, showing that 0RS020607 moved between treatment plots at both sites. However, at both sites, nodules from plots of trees that were inoculated originally with local crushed nodules gave reactions in ELISA that were significantly lower than values for 0RS020607 inoculated trees, possibly due to the competitive effects for new nodulation of enhancement of the indigenous population of Frankia. Serological techniques using antibodies of high specificity against Frankia strains have value for rapid screening of field samples as a preliminary for further analysis by more discriminatory techniques based on assays of genetic polymorphisms.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Host range ofFrankia endophytes

Summary Cross-inoculation experiments with 10 pure cultured strains and 17 host species were carried out. The 10 strains were isolated from the root nodules on actinorhizal trees ranging in 9 species, 5 genera and 4 families. The host species belong to 5 genera. The pure cultured strains fromAlnus are of strong ability to infect different species of the same genus. The seedlings inoculated with these strains are able to nodulate normally. These strains can also infect and nodulate the seedlings ofMyrica californica, but not the seedlings of Elaeagnus, Casuarina andMyrica rubra. The pure cultured strains from Elaeagnus can infect and nodulate the host species in the same genus and family with an exception ofE. viridis vardelavayi, which can be only poorly nodulated by a few strains from Elaeagnus. The strains from Elaeagnus cannot infect the seedlings of Alnus andMyrica rubra. The results presented here suggest thatFrankia endophytes can be divided into two groups: Alnus group and Elaeagnus group.     

PCR-amplified 16S rRNA sequence analysis to confirm nodulation of Datisca cannabina L. by the endophyte of Coriaria nepalensis Wall

Different Frankia strains and crushed nodule suspensions were tested for their ability to nodulate Coriaria nepalensis and Datisca cannabina. Datisca cannabina seedlings were nodulated effectively by both crushed nodule suspension from Coriaria nepalensis and Datisca cannabina. The origin of the endophyte in Datisca nodules induced by crushed nodules of Coriaria was confirmed by comparing partial PCR-amplified 16S rRNA sequences with those of the endophytes of both plants. Coriaria seedlings could only be nodulated by crushed nodule suspensions of Coriaria nepalensis. All pure cultures of Frankia used as a single inoculum source or in combinations with a nodule filtrate, failed to induce nodulation on Coriaria. Two atypical Frankia strains Cn3 and Cn7 isolated from Coriaria nodules showed no acetylene reduction activity and did not induce nodulation on the host seedlings.     

Genetic diversity amongFrankia isolated from Casuarina nodules

The nodulation ability of variousFrankia strains isolated from the nodules of Casuarina were tested on two Casuarina species (C. equisetifolia andC. glauca), and onHippophaë rhamnoides. We found that the isolates could be separated into two groups, some of them being unable to reinfect the Casuarina host-plant but infective onH. rhamnoides. Other isolates effectively nodulated the original Casuarina host-plant. The second purpose of this study was to examine the genetic diversity among the Casuarina-isolated strains using well-characterized symbiotic genes as hybridization probes. We found a relationship between nodulation characteristics and hybridization patterns.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.     

The influence of fertilizers on root rot of field peas caused by Fusarium oxysporum,Pythium vexans and Rhizoctonia solani inoculated singly or in combination

A Frankia strain ISU 0224887 was isolated from spore negative root nodules of Gymnostoma sumatranum and was grown in pure culture. It was infective and effective for Gymnostoma species but failed to nodulate Allocasuarina and Casuarina seedlings. Light and scanning electron microscopy of it in nitrogen free medium revealed a filamentous mat of septate and branched hyphae bearing sporangia and vesicles capable of fixing nitrogen. The strain also produced an orange pigment after 2 weeks culture. The strain utilized only TWEEN 80 and propionate as sole carbon sources. The different antibiotics used showed varying effects on its growth.     

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.     

Nodulation patterns of actinorhizal plants in the family rosaceae

Patterns of nodulation, growth, andFrankia — host specificity have not been well characterized for the actinorhizal genera in the family Rosaceae because of the scarcity ofFrankia isolates from these taxa. Furthermore, the few isolates available from actinorhizal Rosaceae have consistently failed to nodulate plants from the host genus. In a series of experiments, species of rosaceousDryas, Cowania, Cercocarpus, Fallugia, andPurshia were inoculated withFrankia isolates, crushedDryas actinorhizae, and neoglacial soils to ascertain whether any of these inocula would effectively induce nodulation. Neoglacial soils from Alaska and Canada nodulated not only the localDryas drummondii, but alsoCercocarpus betuloides, Cowania mexicana andPurshia tridentata from distant and ecologically diverse locales as well as nonrosaceous, actinorhizal species ofAlnus, Elaeagnus, Myrica, andShepherdia. But of eightFrankia isolates, including two fromPurshia tridentata and one fromCowania mexicana, none were able to induce nodulation onPurshia orCowania species. Globular, actinorhizae-like nodules incapable of acetylene reduction were produced onC. betuloides inoculated withFrankia isolates. Crushed nodule suspensions fromDryas drummondii nodulated rosaceousCowania, Dryas andPurshia, as well as non-rosaceousElaeagnus, Myrica, andShepherdia species. Nodules produced by inoculation ofCowania mexicana andPurshia tridentata with crushed, dried nodule suspensions fromDryas drummondii reduced acetylene to ethylene, indicating nitrogenase activity for these nodulated plants. These data suggest that a similar microsymbiont infects the actinorhizal genera in the family Rosaceae.